Xerosin and process of preparation thereof



June 19, 1962 V. GROUP ETAL XEROSIN AND PROCESS OF PREPARATION THEREOF'Filed Nov. 20, 1956 5 Sheets-Sheet 1 WAI/E LEA/6TH TnrL JNVENToRsI//A/CE/V GROUPE fa/WHA H. P06/l BY ALW/v s. Ew/v5 `lune 19, 1962 Ava.wr aA/5s (au) XEROSIN AND PROCESS OF PREPARATION THEREOF Filed Nov. 20,1956 5 Sheets-Sheet 2 O- 00A/7120i l l I l l l l l l l l 1 2 3 4 5 6 '7,K4/ZM Arran/5y June 19, 1962 v. GROUPE ETAL 3,039,923

xERosIN AND PRocEss oF PREPARATION THEREOF 0 I l l l l June 19, 1962 v.GROUPE ETAL XEROSIN AND PROCESS OF PREPARATION THEREOF 5 Sheets-Sheet 4Filed NOV. 20, 1956 June 19, 1962 v. GROUPE: ETAL 3,039,923

xERosIN AND PRocEss oF PREPARATION THEREOF Unite States Patent Y satan?.Patented June 19, 1962 The present invention relates to a microbialproduct, xerosin, which is active as an anti-inflammatory agent and iscapable of beneficially affecting certain viral diseases, and a methodof preparation of such product.

The present invention is a continuation-in-part of our prior andcopending application Ser. No. 381,268, filed September 21, 1953, nowabandoned, and entitled Microbial Product Capable of BeneficiallyAffecting Certain Viral Diseases and Method of Preparation Thereof.

In the past, there has been considerable activity in the development ofpharmaceutical, and chemotherapeutic preparations for the therapeutictreatment of various diseases, some of which are caused by microbesincluding the bacteria, rickettsiae and viruses. is known, the efforthas been, in substantially all cases, to provide a material usable ortolerated in the body of the animal or human, as the case may be, whichwillV either kill the bacteria or virus (a bactericidal, or virucidalagent) or which will prevent the multiplication of the bacteria or virus(a bacteriostatic or virustatic agent) in some way in the body of thehost. Many of the present-ly known chemotherapeutic agents such, forexample, as the sulfa-drugs, penicillin and streptomycin are in thislatter class of agents, i.e., these substances tend to preventmultiplication or reproduction of the living organisms Which cause thedisease in question, and hence permit the defense mechanisms of the host(such as phagacytosis and antibody formation) to rid itself of theparasites.

There is a theory, which is presently believed to be correct and whichis supported by a progressively increasing volume of evidence, to theeffect that the multiplication of viruses, per se is, in certaininfections, independent of the progress of the disease or lesion itself.It is believed true that the inception of the disease in a host requiresa certain threshold number or concentration of the infectious virus.However, once this threshold amount or concentration is reached and thedisease or lesion has its inception, the number of virus particles mayactually be on the decrease, while the disease continues to develop orincrease, even to the point of causing the death of the host. For thisreason, therefore, it is believed that the previously known antiviralagents, which act either as virucidal or virustatic agents, may beineffective, per se, to accomplish a desired complete therapeutictreatment, at least in certain instances.

Xerosin is believed to be an answer to the need just pointed out, inthat it is an anti-irdiarnmatoryV agent which tends to reduce orsuppress inflammation, or in some instances to reduce or suppress thegrowth of certain types of tumors as hereinafter set out in detail, eventhough xerosin does not itself serve to kill or even inhibit the growthof the bacteria or virus causing the lesions or inflammation. Xerosinhas further proven effective in reducing or suppressing inammation ininstances where the inliammation was caused by purely chemical means inthe absence of any bacteria or virus.

From another point of View, it now seems probable that lesions and/orinammation may be caused either by the direct action of bacteria orvirus or similar direct action of certain toxic chemical agents, or maybe caused as a consequence of the reaction of the host to direct orindirect action of bacteria, virus, or toxic chemical agents. in anyevent, whatever be the cause of the lesions or inliammation or Whateverbe the mechanism by which it is brought into being, it is found that inthe cases of many lesions and types of inflammation including for As faras.,

example, certain types of tumors as hereinafter set out,

the action of xerosin in suppressing the growth or spread of suchlesions, inflammation or tumors and in reducing or minimizing theirundesirable character by general beneticially affecting them, has beenproven and is a present known characteristic of xerosin. it has beenshown that xerosin does not itself, apparently either directly orindirectly, kill or prevent the multiplication of either bacteria orvirus as far as is known, notwithstanding its beneficial eect uponlesions and upon inflammation which may have been produced by the director indirect action of to the direct and/ or indirect action of certainrepresentative viruses as particularly hereinafter set forth. There isalso presented herein a process and general teachings as to the mannerin which this new material, xerosin, may be prepared, and, to someextent at least, rened or purified.

Considering briefly the characteristics of xerosin, it is a material,which is believed to be organic in character as distinguished frominorganic; which contains both nitrogen and phosphorous, each in theform of one or more of its compounds; which is not dialyzable againstrunning water; which isquite soluble in water and also in dilute aqueousalkaline solutions; which is quite thermostable; which is substantiallyinsoluble in many common organic solvents, such as ethanol; which issubstantially ineffective in vitro against representative fungi,bacteria, bacteriophages and animal viruses; which is ineffective invivo as a virucidal or virustatic agent; which responds as follows tocertain color tests:

Biuret Positive Anthrone Positive Iodine Negative Molischs NegativeFehlings Negative Millons rement Negative Nitroprusside NegativeNinhydrin Negative which has a characteristic absorption pattern forultraviolet light as hereinafter set forth; which is elective in vivo insuppressing the development of the disease or lesion in mice resultingfrom previous infection with any one of the following representativeviruses:

influenza A virus influenza B virus v Mouse pneumonitis virus (Miyagawanella bronchopneumonae) Newcastle disease virus (NDV) 3atmospheric oxygen, and precipitating'crude Xerosin from the liquidculture by acidifying it to a pH of about 2 to `about 4, and preferablyabout 3.5, and further refining or purifying the crude Xerosin thusprecipitated by repeatedly, redissolving the precipitate in a neutral orslightly yalkaline medium and reprecipitating by acidification withinthe limits and/ or in the preferred acid concentration hereinabovenoted. If desired, a further purification procedure may be employed,including redissolving the material and reprecipitation thereof from 50%ethanol containing one percent sodium chloride, preferably, but

Vnotnecessarlly, in the cold, at about 6-9 C.

The present invention will be set forth in greater detail hereinafter asto each step of the preparation thereof, the tests which have been madeof the material itself and its chemical and physical characteristics,and also, and particularly, its effect in suppressing the development ofthe disease or lesionitself. This subject matter will be betterunderstood by reference to the accompanying drawings in which:

FIGURE l is a chart illustrating the ultraviolet absorption of Xerosinplotted against the wave length (APM in FIGS. l-lO is Xerosin);

FIG. 2 is a composite chart indicating the development of pneumonia inmice, including an upper portion wherein the ordinates are in units ofpercent pneumonia, the'values plotted being based in each instance uponthe average of a number of visual observations; a lower portion whereinthe ordinates are in units of the average weight of lungs; and in bothportions, the abscissae are days after inoculation of the mice withvirus; curve I in both lower and upper portions representing a control,and curve II in each portion representing the effect of daily injectionsof Xerosin, beginning' 1 hour after the instillation o f NDV (Newcastledisease virus);

FIG-3 is a composite chart similar to FIG. 2 described above, exceptthat in each portion of the chart, curve I is the control; curve IIrepresents the result of 2 daily injections of Xerosin beginning l hourafter instillation of NDV; and curve III shows the similar effects ofdaily injections of xerosin for 3 days;

FIG. 4 is a similar composite chart illustrating the eect of a dailyadministration of Xerosin on `the development of pneumonia, curve I ineach instance being the control; curve Il illustrating the effect ofdaily injections of xerosin, ybeginning 48 hours after the instillationof NDV; and curve III illustrating the eiect of daily injections ofXerosin beginning l hour after instillation of.

NDV;

FIG. 5 is a chart illustrating the infective titer of infected lungtissue, the ordinates representing the log of dilution downtoy the pointwhere the diluted tissue will be effective only to infect one-half or50% of the animals subjected thereto, while the abscissa is days afterthe inoculation; curve I representing the results aforesaid whereinXerosin is injected daily beginning l hour after instillation of NDV,and curve II being the control;

FIG. 6 is a chart of percent pneumonia based on data obtained asdescribed in connection with FIG. 2, against the reciprocal of thedilution of NDV, curve I representing the control and curve Il, theeffect of daily injections of Xerosin beginning l hour after theinstillation of NDV;

FIG. 7 is a composite chart similar to FIG. 2, illustrating the effectof Xerosin on mice infected with mouse pneumonitis virus, curve Irepresents the control and curve II illustrates the effect when Xerosinis administered at the rate of 3 mg. per day, beginning the third dayafter infection;

FIG. 8 is a composite chart similar to FIG. 2, illustrating the elfectof l mg./ day Xerosin alone administered subcutaneously,chlortetracycline alone and combined therapy using both Xerosin andchlortetracyclne, all tests being in mice, infected with mousepneumonitis virus;

FIG. 9 is a composite chart similar to FIG. S above described, exceptthat 3 mg. per day of xerosin were ernepee 4, ployed in the testsreported in this gure as contrasted with l mg. per day for the testsillustrated in FIG. 8; and

FIG. 10 is a chart similar to FIGS. 8 and 9 except that in this case theordinates are in the terms of cumulativepercent mortality, while theabscissae is in the terms of days as in FEGS. S'and 9, this figureillustrating the effect on mice infected with mouse pneumonitis virus,of the separate and combined use of Xerosin and chloramphenicol. Y

In the detailed specification which follows, the subject matter will beconsidered under several headings for the convenience of the reader.

THE ORGANISM WHICH PRODUCE/S XEROSIN The bacterium which is used toproduce Xersoin in accordance with the present invention was isolatedfrom soil. This bacterium is known as 'Achromobacter xerosz's No. 134.This species of bacterium is a new species, and has been namedaccordingly.

The bacteria or cells consist essentially of rods, usually about 3.5/1.,by 2p. to 3p. in liquid cultures after twentyfour hours of incubation at28 C. Cells as long as l0 to 25u are not uncommon in similar culturesincubated for forty-eight to seventy-two hours. The cells are motile bymeans of peritrichous flagella. The cells occurred singly or in shortchains. Capsule formation was not observed. Grams reaction was negative.The cells or rods are non-acidfast, and non-spore-forming.

On nutrient agar colonies were formed measuring 1.0 to 1.5 mm. indiameter and were White or greyish white in appearance. T'he colonieswere dry, membranous, circular with Van even edge, low convex and wereadherent to the surface of the agar. On prolonged incubation, thecolonies became tan in color, somewhat granular,

and radially wrinkled with a lobated edge.

The cultures grew well at temperatures of 28 and 37 C. Cultures of thisbacterium may be grown so as to produce xerosin in liquid mediacontaining complex nitrogenous materials, such, for example, as peptone.In stationary culturing Xerosin Was produced and a pellicle was formedin broth containing any of the following: peptone, proteose peptone,tryptose and tryptone.

rI'his bacterium may also be cultured and grown in an agitated bath or amoving bath, except that under such conditions, no pellicle is formed.Gaseousoxygen is, however, required in all culturing methods, as theculture is aerobic and growth apparently does not occur in the absenceof gaseous oxygen.

The culture of the Achromobncter xerosis No. 134 may also be grown, butwithout producing Xerosin or anything equivalent thereto, in'mediaincluding inorganic nitrogen, such as an aqueous solution of dibasicammoniurn phosphate [(NH4)2HPO4] as the sole source of nitrogen. Thisbacterium will grow, but will not produce an acid reaction, in nutrientbroth containing the sugars: glucose, galactose or maltose. On the otherhand, on a dibasic ammonium phosphate-base agar, `an acid reaction wasproduced when the medium contained glucose, galactose o-r maltose vasthe sole source of carbon. There was some growth without production ofan acid reaction on agar containing lsucrose as the sole Source ofVcarbon. There was no growth on 4agar containing lactose, arabinose,sorbitol, Xylose, rhamnose, salicin, mannitol, or raliinose as the solesource of carbon.

VThere was no growth on initial transfer to inorganic nitrogen base agarcontaining 50 img. per l0() ml. of phenol. The `growth of this bacterium4on other direrential media is as follows:

(a) Litmus milk became alkaline and litmus was reduced after 7 daysincubation.

(b) Starch was hydrolyzed.

(c) Gelatin was liquifled.

(d) Indol was not produced from tryptophane.

(e) Citrate was utilized as the sole source of carbon.

(f) Some hydrogen sulfide was of incubation.

(g) Growth on potato was yellowish to brownish and -appeared dry andwrinkled.

A culture of Achomobacter xeross No. 134 has been deposited in theculture collection of the Institute of Microbiology of Rutgers, theState University, New Brunswick, yNew Jersey, under the number, 134.

THE PREPARATION OF XEROSIN Considered broadly, xerosin is produced byculturing the producing bacterium described above, i.e., Achromobacterxerosis, No. 134; then ltering the culture medium (a step usually neededat this stage, although it may be replaced by a Lstep involvingseparation of undesired solids at some later stage); then precipitatingthe crude xerosin from the filtrate by adjusting the pH of the ltrate toa value from about 2 to about 4, and preferably about 3.5; thenpurifying the crude xerosin by repeated solution in an aqueous mediumhaving a pH greater than about 4 and reprecipitation by adjusting the pHof the resulting solution to an acid condition of about 2 to about 4 andagain preferably about 3.50.

The culture medium in which the producing'bacterium, Achromobacterxerosis No. 134, is placed to lgrow and to produce xerosin shouldcontain some complex nitrogenous medium, such as peptone. On the otherhand, peptone, per se is not required, nor is the character of themedium restricted to any small number of dierent materials. Asaforesaid, the bacteria may be cultured in a stationary medium toproduce a pellicle, wherein the nutrient medium comprises a brothcontaining peptone, tryptose, or tryptone. Other media, usually protem-containing in nature, such as proteose peptone, or other brothsformed from meat, generally containing complex nitrogenous materials,such as protein, may be used as the culture medium. Vegetable proteinsor analogous materials may also serve as the culture medium. In fact,there are so many things which could be used, only a relatively few ofwhich have been experimented with, that the composition of the culturemedium should not be considered as a narrowly critical factor from anypoint of view.

`One specific culture medium, which has been found to be not only fullyoperative, but also desirable in the culturing of `this bacterium in theproduction f Xerosin, has the following particular composition:

produced after 7 days `Percent Glucose 0.25 Yeast extract 0.1 Peptone1.0 Beef extract 0.5 Distilled or tap water Balance cable to the othertypes.

-In culturing, it may be desirable to prepa-re an inoculum pool from anactively growing culture of Achromo bacter xerosz's No. 134 in a culturemedium, having the particular composition listed above, after 24 hoursof incubation at 28 C. To this is added 20% sterile inactivated horseserum, before distribution of the resulting material into a suitablenumber of vials. These vials are then stored at 70 C. until used or maybe dried from the frozen state.

It has been found that agar slants of Achromobacter xerosz's No. 134were found to lose their viability after 7-14 days storage in therefrigerator. However, in each of several experiments, frequentlytransferred broth (of the composition listed above) cultures were stableat refrigerator temperatures and xerosin production was alwayssatisfactory and occasionally was greater. Frozen inocula -were employedonly as a convenience and to avoid possible variation or lysogenicity inthe culture. Lysogenicity has never been observed here.

Blake bottles, each containing 250 ml. of sterile nutrient medium, thecomposition of which was listed above, were each inoculated with 5 ml.of seed, this seed consisting of the third serial transfer from thefrozen inoculum, the preparation of which is described above. A smear ofthe seed was examined microscopically before each transfer. Theinoculated Blake bottles were incubated horizontally for 3 days at 28 C.A surface pellicle was formed by the second day and the nal reaction ofthe vmedium was pH about 7.5 to about 8.5.

Xerosin has also been prepared using shaken or submerged cultures,Iwhich were incubated for 72 hours at 28 C. It seems probable thatsomewhat shorter periods of incubation would also be effective. In anyevent, care must be taken, irrespective of the mode of culture, toassure that the culture has access to gaseous oxygen. One way ofaccomplishing this in the Blake bottles is to have the bottles closedwith an air-pervious closure, such as non-absorbent cotton. In largescale operations, air may be caused to bubble up through the culturemedium during the incubation period, thereby simultaneously eecting bothaeration and agitation.

Returning now to the method of culturing the bacterium in question on astatic basis and the subsequent procedure to recover xerosin therefromand to purify the crude xerosin recovered, it is found that a pellicleis formed by the second day of the incubation. This pellicle ispreferably removed by ltering the culture products through a suitablelter medium such as gauze or glass wool, the pellicle (which consistsentirely of bacterial cells) being discarded. The filtrate is thenacidiiied with concentrated HCl to a pH of about 2 to about 4, andpreferably about 3.5. The crude xerosin, which is precipitated, isallowed to settle by gravity and/or may be separated from thesupernatant liquor, either by decantation or ltration. lt has :beenfound desirable at this stage of the process to remove the supernatantliquor by suction, using a water pump. The precipitate of crude xerosinmay then be repeatedly dissolved and reprecipitated, for example, ineach instance, by dissolving the precipitate in about l5 volumes ofdistilled water, neutralized to a pH of 7-10 with 5 N NaOH.

The cycle of precipitation by acid, subsequent dilution andneutralization may be repeated any desired number of times. For example,three such additional times have been found to be satisfactory for manypurposes. On the fourth dilution, 3 volumes of distilled water wereadded and the solution was adjusted to -pH 9.0-9.5. This solution wasclarified by centrifugation using a Sharples super centrifuge (24,000r.p.m. at a slow rate of ow). The sediment was discarded and thesupernate (eflluent) was diluted with 5 volumes of distilled water andagain acidiiied with HC1 to pH 3.50 to precipitate xerosin. After' theprecipitate settled by gravity, the supernate was discarded, and a finaldilution of 3-5 volumes of distilled water and acidification to pH 3.50was carried out. The final precipitate was neutralized (pH 7.0-7.5 in aminimal volume of distilled water and was then dried from the yfrozenstate. Xerosin was stored in a desiccator under vacuum. This method hasbeen found to yield -200 mg. of xerosin per liter of the originalculture filtrate treated.

vlt has been found that the best results in precipitating xerosin and inthe settling of the precipitate are attained at a pH of about 3.50. Ithas also been found that xerosin is substantially insoluble in anaqueous solution having a pH from about 2 to about 4. This pH range is,therefore, to be considered fully operative throughout with thepreferred value, particularly for the re-precipitations at a pH of about3.50.

When the culturing is eiected in a shaken culture, the same methodsgenerally described above, `may be ernployed with the following changes:

(1) Preliminary filtration through gauze for removing the pellicle whichis produced during static culturing, is

unnecessary;

(2,) An additional clarification by centrifugation may be introducedafter neutralization of the first acid precipitate. At this stage thevolume to be centrifuged was about /o that of the original culture.

The Xerosin material produced and purified as above set forth may, ifdesired, be further purified, without apparent loss of any activeprincipal or ingredient of the Xerosin, but with improved results as topotency and lack of toxicity by a precipitation following those abovedescribed in 50% ethanol containing about 1% sodium chloride. Thisprecipitation is preferably, but not necessarily, carried out at arelatively low temperature (under refrigeration) at about 6 to 9 C. Thepurified Xerosin thus produced is a white solid, which is highly solublein Water, and which has characteristics which will be described in thesucceeding section.

The following comments may be made in respect toV PHYSICAL AND CHEMICALPROPERTES OF XEROSN Xerosin, as above set forth, is a substantiallywhite solid material which, due to its being dried by evaporation from afrozen slurry, has a low bulk density. In a somewhat impure form, it maybe light tan in color. This material is -very soluble in distilled wateras well as being soluble in dilute alkaline aqueous solutions. It isalso reasonably soluble (e.g., 1 mg. per ml.) in physiological saline.

Xerosin is substantially insoluble in many common organic solvents such,for example, as ethanol, n-butanol, anesthetic ether, chloroform,petroleum ether, ethyl acetate, benzene and acetone. The potency ofXerosin in aqueous `solution (distilled water) is apparently notaffected by any of the following: (a) exposure at 100 C. at either pH 6or pH 9 for tive minutes; (b) exposure in an autoclave at 120 C. fortwenty minutes; (c) eX- posure to liquid ethylene oxide for one hour atC.; (d) precipitation with 90% ethanol; (e) dialysis against running tapwater for 24 hours; (f) precipitation from aqueous solution by halfsaturation with ammonium sulfate; or (g) precipitation from aqueoussolution'by addition of about 9 volumes of ethanol.

Various lots of Xerosin have been analyzed chemically to determine thenitrogen and phosphorus content thereof. The average of thesedeterminations show a nitrogen content of about by weight (based onXerosin corrected to a zero moisture content) and phosphorus content ofabout 2% by weight. There is apparently a zero sulphur content as far ascan be determined.

While it is not known whether Xerosin is one single chemical compound ora mixture of two or more chemical compounds, the percentage of nitrogenand phosphorus therein plus its reaction to certain color tests as ter.

consist of or contain some matter similar to nucleic acid.V

hereinafter noted would seem to indicate that it may consist of orcontain a material of polypeptide charac- It'is also possible that thismaterial Xerosin may The material (Xerosin) has been tested as to itsabsorption of ultraviolet light. The results of these tests are shown inFlG. 1, wherein a peak at 255-260 mu is positively shown.

The following is a report of the exposure of a nurnber of the samples ofxerosin, each made as aforesaid, to various color tests:

(1) Fehlings solution or Benedicts reagent: None of the samples testedgave any significant reduction.

(2) Molischs reagent: Solutions of some samples of Xerosin gave veryfaint violet rings with traces of green coloration in the sulfuric acid.The test was interpreted as negative.

(3) Anthrone test: Addition of the concentrated acid and anthroneresulted in an immediate and typical color change indicating thepresence of carbohydrate, which was apparently tightly held since theMolisch test (see 2. above) was essentially negative.

(4) Millons reagent: This reagent caused precipitation of the material.This test was essentially negative, although phenolic residues may bepresent.

(5) Nitroprusside test (cysteine): This reagent gave no coloration withany of the samples tested. The absence of sulfur Was shown bycombustion.

(6) When various samples were tested with I2 solution, no coloration wasobserved. N

(7) Ninhydrin reagent: The results were negative with all samplestested.

(8) Biuret test: Each of a number of samples gave positive tests.

(9) A sample of Xerosin was hydrolyzed with HCl and the resultingsolution run on a one dimensional paper chromatogram. A number ofninhydrin-positive spots were observed in confirmation of the positivebiuret test (see 8 above).

After preparation, this material (Xerosin) may be stored at roomtemperature or at some lower temperature, but, because the xerosin isslightly hygroscopic it is preferably stored in a desiccator undervacuum.

BlOLOGlCAL AND THERAPEUTIC IPROPERTLS AND TESTS OF XEROSIN The Xerosin,which was subjected to numerous tests in accordance with this section ofthe present specification, was made in accordance with the methodshereinabove described and was purified through at least fourreprecipitations in acid aqueous solutionas hereinabove explained. Thismaterial has been tested in a considerable number of different wayswhich are hereinafter described.

When tested in vitro, no evidence of antibacterial or antifungalactivity could be demonstrated when the streakdilution method wasemployed. A concentration of 5 mg. of Xerosin per ml. in nutrient agardid not inhibit the growth of Escherichia coli, M icrococcus pyogenesvar. aureus, Pseudomonas aeruginosa, Shigella sonnei, Bacillus cereus,Bacillus subtilis, ll/Iycobaczerium tuberculosis (strain 607),Streptomycesgriseus, Strepromyces fradiae, Candida albicans, Aspergillusniger or Pencillum nommm.

Xerosin and the culture filtrate from which it can be precipitated werefurther tested in vitro against influenza A and B viruses (separatetests). These particular tests related to the failure of the Xerosin toinhibit hernagglutination (agglutination of chicken red blood cells byiniiuenza virus) ing manner: Infected allantoic fluids were titrated inthe usual manner in the presence of culture filtrate, Xerosin (2 mg. perml.), broth and saline, respectively. The hemagglutinin-test materialmixtures were incubated for one hour at 36 C. before the addition ofchicken The test was carried out in the follow- 9 lerythrocytes.Identical end points were obtained in each of the various titrationswith influenza A and B viruses, respectively. i

In a further test, no evidence of inactivation of Newcastle diseasevirus (NDV) in vitro could be demonstrated when the virus was titratedin the presence of saline, culture filtrate, or Xerosin, respectively,before inoculation into embryonated eggs.

Considerable time and effort were spent in attempting to demonstrateantiviral activity against influenza A virus in embryonated eggs both invitro and in vivo using both culture ltrate and xerosin. Suppressiveeffects, though regularly observed, were minimal. The variouspreparations were rendered suitable for injection into eggs by theaddition of penicillin (500 units per ml.) and neomycin (200 units permL). Control inocula were similarly treated. The results of severaltypical experiments are summarized in Table I which follows:

Table l EFFECT OF CULTURE FILTRATE AND XEROSIN ON INFLUENZA A VIRUS INEGGS i Reciprocal of avg. hemagglutiuin titer of allantoic fluidcollected li9l1rs after infection with l100 IDM. (IDsO is the infectiousdose which will imect 50% of the animals exposed thereto or inoculatedtherewith. 10 ID50 is 10 times this infectious dose, etc.)

2 sterilized with ethylene oxide.

Nora-Contact test equals test material mired in vitro with virus andincubated l hr. at room temperature before inoculation. Therapeutic testequals test material injected 1 hr. after infection. Protection testequals test material injected l hr. before injection. I/T equals thenumber of mice infected over the total number.

' The data indicate that 4culture filtrate known to be etective insuppressing the development of pulmonary lesions in mice, was, at best,capable of effecting a slight (approximately 2-fold) reduction in theaverage hemagglutinin titer of allantoic fluid collected 48 hours afterinfection with -100 ID50 of influenza A virus. However, this slightreduction in the formation of viral hemagglutinin was obtained whetherthe culture liltrate was mixed in vitro with the virus or was injectedone hour before or one hour after infection. It was hoped that Xerosinwould prove to be more effective .than culture ltrate. However, this wasnot the case. A solution of xerosin `containing 5 mg. per ml. wassterilized with ethylene oxide, and was tested for antiviral activity bymeans of the contact test. As with culture filtrate a slight(approximately 2-fold) reduction in viral hemag- :glutinin was obtained.Attempts to demonstrate a reduction in the infective titer of allantoicfluid collected 24 hours after inoculation were unsuccessful. The eggSreceived potent culture filtrate one hour after infection with l0 IDEOof virus. It was concluded that the embryonated egg was not a suitablehost for the demonstration or detection of antiviral activity of xerosinagainst iniiuenza A virus when the usual methods were ernployed.

Many tests were conducted using white mice as the host and using variousviruses. The first series of tests in this group hereinafter set forthall used inuenza A virus.

Data from two typical experiments illustrating the suppressive effect ofxerosin on the development of pneu- T able Il SUPPRESSIVE EFFECT OFXE-ROSIN ON PULMONARY LESIONS IN MICE INFECTED WITH INFLUENZA A VIRUSResult-3rd day after infection 1 Mg. xerosin injected C on day Lesionscore Avg. I/T weight lungs, g. 0 l 2 L/M 0 0 0 21/22 48/105 46 9 9 9/l2 4/60 7 3 3 3 12/12 8/60 13 1 l 1 6/10 5/50 l0 3 3 3 10/12 10/6() 17l l l 11/12 21/60 35 z0/6 0 0 0 15/15 53/75 7l 3 3 3 5/12 7/60 l2 0 3 312/12 20/50 33 0 O 3 12/12 28/60 47 0 3 0 11/12 19/60 32 3 3 0 10/1220/60 33 3 0 0 S/12 14/60 23 1 Approximately 10,000 1D5o. 2 Uninfectedcontrols. Y

No'rE.-SC=subcutaneously. I/T=Nun1ber of mice infected/total LlM=Totallesion score/total max. score. %=L:MX 100.

Lesion score: 5% lung tissue consolidated; l=5-25%; 2=2650%; 3=51-75%;4=7-lO0%; 5= dead mouse with lungs consolidated.

Groups of 12 or more mice each Were infected intrana-` sally withapproximately 10,000 ID50 of iniiuenza A virus `and injections ofxerosin were begun one hour or more after infection as indicated. Allmice were sacriiied on the 3rd day after infection and the degree ofpulmonary consolidation and average weight of the lungs were recorded.The data indicate that (a) daily injections of Xerosin were thel mosteffective, (b) a delay of 24 hours in the time of treatment or areduction in the number of injections of Xerosin reduced itseffectiveness, (c) a suppressive effect was still demonstrable when onlyone injection of xerosin was given 48 hours after infection, and (d)daily injection of as little -as 0.3 mg. of xerosin exerted a detectablesuppressive effect on the devel0pment of pulmonary lesions. It is ofinterest in this connection to recall that the infective titer of lungtissue has been shown to be maximal 24 hours after infection with lowdilutions of influenza A virus.

Comparative studies on the relative effectiveness of Xerosinadministered by various routes were carried out in similar experimentswith influenza A virus. The data obtained indicated that intraperitonealinjection ofxerosin toxic for mice; and that in 10-12 gram mice, themaximum tolerated dose (LDU) is about 800 mg. per kilogramsubcutaneously, 400 rug/kg. intraperitoneally and 200 mg./kg.intravenously. inasmuch as xerosin is not eective when administeredorally, the maximum tolerated-dose for oral administration has not beendetermined. As compared with these figures, however, the averageeffective dose in mice are daily injections of 50-150 mg. per kilogramof ybody weight. At this level, it is believed that a considerableexcess is being administered and is apparently far below the toxiclevel.

`Intranasal instillation of xerosin virus mixtures was also found to beVineffective in the following experiment: One ml. amounts of serialdecimal dilutions of influenza A virus were mixed in vitro with an equalvolume of saline containing 3 mg. of Xerosin per m1. respectively, andincubated for one hour at room temperature kbefore inoculation intogroups of 5 mice each day by the intranasal route. The mice wereobserved for a period of 10 days and dead mice and mice still living onthe 10th day after inoculation were examined and the degree of typicalpulmonary consolidation was recorded. 'Ille end acariens 1i points(i950) of the 2 titrations were identical (l0-H), clearly indicatingthat xerosin Was not virucidal for inuenza A virus. i

The term titrations, as used herein, indicates the degree of dilution ofthe infectious material down to the point of the so-called end point,i.e., Where the dilution is such as to infect only 50% of the animals towhich a material in this degree of dilution is administered. It isusually expressed as a negative power of l0.

It was of interest to determine Whether xerosin Was more eiective whenmice were infected with a much smaller quantity of inuenza A virus. Inthe experiment summarized in Table El, shown below, groups of 20 miceeach Were infected with a sublethal dose [l0 IDM] of virus and wereinjected daily, subcutaneously with l and 3 mg. of xerosin,respectively, beginning one hour after infection.

Table 111 EFFECT OF XEROSIN ON DEGREE OF PLMONARY CON- SOLIDATION INhICE INFECTED YVITH SUBLETHAL DOSE OF INFLUENZA A. VIRUS Result-th da;7after infection,l lesion score Twenty similarly infected, but untreated,mice served as the control group. All mice Were sacriiced on the 10thday after infection and the degree Vof pulmonary consolidation wasnoted. The data indicate that daily injections of xerosin definitelysuppressed the development of pulmonary lesions'in mice infected with asublethal dose of iniiuenza A virus. However, it Would appear that theetlectiveness of xerosin in suppressing the development of pneumonia Wasnot markedly increased when the size of the infecting dose of virus wasgreatly (LOOOX) reduced (Table il).

'It was of obvious importance to study the eect of xerosin on the rateof death of mice infected with a lethal dose of influenza A virus. Theresults of a typical experiment are summarized in Table IV whichfollows:

Table 1V EFFECT OF XEROSIN ON RATE OF DEATH OF B/IICE IN- FECTED NVITH10,000 IDsn OF INFLUENZA A VIRUS This experiment was identical With thepreceding one except that a lethal dose of virus (10,000 ID50) wasemployed. The data show that daily injections of 3 mg. of xerosindelayed death of the mice by approximately 2 days and that when thedaily dose of xerosin injected ywas decreased to l mg., the delay indeath was reduced.

A series of tests substantially similar to those above described andherein reported as to influenza A Virus in the mouse were also carriedout using inuenza B virus in the mouse. The results Were substantiallythe same as `those hereinabove reported as to inuenza A virus. For thisreason, these particular data are not included herein.

The tests reported above, which Were carried out using inuenza A virusand influenza B virus, related to the use of a virus capable ofmultiplying, both in its inherent character and also in view of theportion of the host in which these viruses Were introduced. Under thesecircumstances, a relatively small amount of virus will be capable inmost instances of inducing the disease or lesion in question due to theprogressive.multiplication of the Virus, raising the concentrationthereof up to the threshold concentration characteristic of theparticular host-virus relationship. When that threshold concentrationobtains, in accordance with the present theory, the disease or lesionbegins to develop. 'Ihis lesion is suppressed, as hereinabove set forth,by the action `of xerosin. However, when the virus is Vof the kindand/0r in such location that it can multiply and continues to multiplyin the host, the effect of xerosin may be only temporary if used alone,as the continued multiplication of the virus and increasingconcentration thereof tends to oiset the beneiicial etiect of xerosinafter a time. It has been v)found that the eiect of xerosin Willsuppress the lesion and retard its development for a period of about 48to about 72 hours.

There are some viruses which produce disease, however, which whenintroduced in low dilution into certain hosts are not capable ofmultiplication, such as Newcastle disease virus (hereinafter referred toas NDV) which is inherently incapable of multiplication to anysubstantial extent in mice. Also, properly classiied in this generalcategory, are viruses which are inherently `capable of multiplication inthe host, but are introduced in such a portion ot the host that viralmultiplication does not occur. This will be treated more in detailhereinafter.

It has `been shown that the non-,transmissible pneumonia in mice, whichfollows intranasal inoculation of NDV, paralleled the reactions betweenvirus and host cell, save that formation of new infectious particles didnot occur. Further, when the maximum of pneumonia was observed, thereremained less than 0.1% of the original NDV inoculum. This uniquehost-virus relationship was found to be affected by xerosin in theexperiments described below.

A large number of mice were inoculated intranasally with 0.1 ml. ofundiluted allantoic fluid containing 109 infectious units of NDV.Beginning one hour after inoculation and daily thereafter' groups of l5mice each were injected subcutaneously with various amount of xerosin asindicated in Tabie V which `follows:

Table V EFFECT OF XEROSIN ON DEVELOPMENT OF PNEUMONIA INDUCED BYNEWCASTLE DISEASE VIRUS IN MICE Result-3rd day after inoc.1

erosiu Lesion score Avg. I/T Weight lungs, g. LIM

2 0/4 IS 30,30 10o/iso 61 27 rai/15 20,175 Y 27 .21 13/15 Z0/75 27 2214/15 19/75 25 22 10/15 18/75 24 23 l2/l5 34/75 45 25 1 109 IDn. 2Uninoculated controls. Y

inoculated but otherwise untreated mice served as controls. All micewere sacrificed on the 3rd day after inoculation (at the time of maximumpneumonia) and the degree of pulmonary consolidation and the averageWeight of the lungs were recorded. It is clear from the data that dailyinjection of as little as 0.03 mg. 'of erosin suppressed `but did notwholly prevent the development of pneumonia.

in another extended series of tests, NDV was used as the virus and themouse as the host in each instance. The particular manner of testing wasas follows: Large numbers of albino mice weighing 18 to 20 g. each wereinoculated intranasally Vunder light ether anesthesia with 0.1 ml. ofundiluted allantoic fluid containing 109 IDN, of NDV. After inoculationof NDV the mice were distributed at random into identical cages, 6 to 8mice per cage. The cages were previously marked as control mice or asmice to be injected with xerosin and with the scheduled date ofsacrifice and examination. Injections of xerosin were madesubcutaneously under the loose slcin on the backs of the mice. Groups ofl to l5 mice each were sacrificed at appropriate daily intervals and thelesion score and average weight of the lungs (Weight of petri dish plusl() lungs less weight of dish after removal of lungs, divided by l0)were recorded.

Suppression of pneumonia was readily eiiected by daily injections ofxerosin. Large numbers of mice were inoculated intranasally with NDV.One half of these received daily subcutaneous injections of 1.() mg.amounts` of xerosin beginning 1 hour after inoculation with NDV. Theremaining mice served as controls. Twelve to mice from each group were-sacriced daily for 6 days and on the 9th day after inoculation. Thelesion score, expressed as percent pneumonia, and average weight of thelungs were determined as described. 'Ihe results are presentedgraphically in FIG. 2. As expected, the maximum of pneumonia was reachedin the control group (curve I) on the third day after inoculation withNDV and severe pneumonia continued for three additional days. By theninth day after inoculation, however, resolution of the pneumonia wasWell underway. It is evident (curve II) that the pneumonia wasconsiderably reduced in those mice which received seven daily injectionsof xerosin.

When daily injections of xerosin were discontinued after the secondinjection, pneumonia continued to develop. In this experiment, 17 groupsof 12 mice each were inoculated with NDV. Six of these groups were setaside and served as controls. The remaining mice received 2 dailyinjections of 1.0 mg. amounts of xerosin beginning l hour afterinoculation with NDV. Of these, 5 groups of 12 mice each received oneadditional injection of xerosin on the following day (i.e., the second`day after inoculation with NDV). Appropriate groups of l2 mice eachwere sacrificed at daily intervals and the lesion score and averageweight of the lungs were recorded. The data, presented in FIG. 3, showthat if daily injections of xerosin were discontinued during the periodof rapid extension of the lesion, pneumonia continued to develop.

When injections of xerosin were delayed until 48 hours after inoculationwith NDV, the development of pneumonia was arrested. Large numbers ofmice were inoculated with NDV and then separated into 13 groups of l2mice each. Five of these groups were set aside as controls and an equalnumber received daily injections of 1.0 mg, amounts for tive days,beginning 1 hour after inoculation. In the three remaining groups, dailyinjections of xerosin were not begun until 48 hours after instillationof NDV. Appropriate groups of mice were sacrificed daily and the lesionscore and average weight of the lungs were noted. 'Ihe data aresummarized in FIG. 4 and show that daily injections of xerosin could bedelayed for as long as 48 hours after inoculation with NDV and stillarrest the development of pneumonia.

However, when daily injections of Xerosin were delayed until the time ofmaximal pneumonia, i.e., 72 hours after inoculation with NDV, xerosinwas Without substantial effect. In this experiment, as before, largenumbers of mice were inoculated intranasally with NDV. One group of 19mice was sacriiiced on the third day after inoculation and the lesionscore and average weight of lungs were recorded. 4A second group of 24mice received daily injections of 1.0 mg. amounts of Xerosin beginningon the third day after inoculation. A third group of 22 mice served ascontrols. On the 7th day after inoculation with NDV, mice in the secondand third groups were sacriced and examined. The data are presented inTable VI, which follows:

Table VI FAILURE OF XEROSIN TO AFFECT RESOLUTION PNEUMONIA IN MICE 1l()9 ID5u intranasally. 2 1 mg. amounts of xerosin injectedsubcutaneously daily beginning on the 3rd day after inoculation of NDV.

NoTE.-L/T=Number infect d/total. LlM=Total lesion score/total maximumlesion score. %=L+M 100.

Lesion score: l= 5% lung tissue consolidated; l=5`25%; 2=26-50%;3=5l75%; 4= 76-10070; 5=dead mouse with lungs consohdated.

It will be seen that xerosin was without substantial effect when dailyinjections were delayed for 72 hours and that it did not affectresolution of pneumonia. Thus, it would appear that xerosin is effectiveonly during the period of rapid extension of the lesion or disease.

Daily injections of xerosin failed to alter appreciably the infectivetiter of lung tissue. Eighty mice were inoculated intranasally with 109IDS@ of NDV. One hour after j inoculation 40 mice were injected-subcutaneously with 1.0 mg. amounts of xerosin and daily thereafter fortwo days. An equal number were set aside as controls. Ten

mice from each group were `sacrificed 2, 24, 48 and 72 hours,respectively, after inoculation. Lungs from the respective groups werepooled and stored at 70 C. Five to eight days later the various pools oflung tissue were thawed, 10% `suspensionsof lung tissue were prepared bygrinding lungs from each subgroup in a mortar with sterile sand, andinfectivity titrations were carried out inembryouated eggs as previouslydescribed. The results are shown in FIG. 5. As expected, the infectivetiter of lung tissue from the control group (curve II) decreasedfrom1084 when removed 2 hours after inoculation to 10-1-s on the third dayafter inoculation. Daily injections of xerosin, which delinitelysuppressed the development of pneumonia, failed to alect appreciably theinfective titer of lung tissue (curve I).

Curiously, dilution of the inoculum of NDV did not increase theeffectiveness of xerosin in suppressing development of pneumonia.Infact, the data indicate an apparent reduction in effectiveness ofXerosin against limiting dilutions of NDV in the experiment describedbelow. Serial 2-fold dilutions of NDV were inoculated intranasally into6 groups of 40 mice each. One hour later half of the mice in each groupwere separated and these mice were injected with 1.0 mg. amounts ofxerosin at that time and daily thereafter for two days. each groupserved as controls. On the third day after inoculation with NDV, whenthe maximum of pneumonia was attained with all dilutions of NDV, ythemice were sacriiiced and the lesion scores were recorded. The resultsare shown in FIG. 6. It will be seen (a) that the effectiveness ofxerosin was not increased when the amount of NDV inoculated wasdecreased; and (b) that xerosin had little eiiect on pneumonia inducedby limiting dilutions of NDV.

When influenza A virus (a Virus inherently capable of growth whenintroduced into portions of the body (i.e., the lungs) of the mousewherein such `growth is permitted) is injected intracerebrally nosubstantial viral multiplication occurs. The virus of influenza A, then,is, in elect, in the same category as NDV. Under these circumstances, arepresentative number of mice were inoculated with influenza A virus andthen given daily injections of xerosin substantially in the same way ashereinabove described in connection with tests on NDV. It was found thatthe daily injections of xerosin substantially delayed The remaining micein and decreased mortality following intracerebral inoculation ofinfluenza A virus.

Tests have also been made in a manner similar to those previouslydescribed herein with mouse pneumonitis virus (Miyagcwanellabronchopeumonz'ae). VXerosil was found to suppress the development ofpneumonia in mice previously infected with this virus. The mode ofaction paralleled closely that previously described for influenza Avirus. In addition it was found that, as in the case of iniluenza A,xerosin did not exert an antiviral effect per se (that is, xerosin didnot inactivate die virus in vitro nor didjit suppressviralmultiplication in vivo); despite this, thedevelopment of pneumoniawas markedly suppressed. This subject matter is illustrated in FIG. 7,which plots the data in the same manner as hereinabove explained inconnection with FIGS. 2 to 4 inclusive.

As above set forth, when a virus is of a kind which is inherentlycapable of multiplication, but is in such a portion of a body of a hostas not to be capable of multiplication under the circumstances, there isstill a possible effect on the host of `a sufficient (threshold amount)concentration of the virus, by reason of the toxicity thereof. Anexample of this is influenza A virus injected intracerebrally into mice.The modifying effect of xerosin on the neurotoxicity of influenza Avirus for truce has been investigated with the following results: Ingeneral this effect of xerosin parallelled the effect previouslydescribed for the suppressive effect of xerosin on the non-transmissiblepneumonia in mice induced by Newcastle disease virus (NDV). As in thelatter case, the influenza A virus does not propagate appreciably, if atall, when injected intracerebrally into mice. However, daily injectionsof xerosin substantially delayed and decreased mortality followingintracerebral injection of iniiuema A virus.

Tests were made of the effect of dailyinjections of xerosin on thetuberculin reaction in guinea pigs. The animals were actively sensitizedwith 0.5 ml. of tubercle bacilli in oil injected subcutaneously 3-5weeks before the test. Five control and live treated animals each weretested by injecting intradermally 0.1 ml. of tuberculin diluted 1/10, l/31.6, and l/l00. The diameters of the areas of induration and erythemaat the sites of injection were observed and measured 24 and 4S hourslater. It was found that xerosin markedly reduced the size of the4tuberculin reactions in sensitive guinea pigs. Necrosis was alsodiminished or absent in these reactions.

Further tests were made of the effect of Xerosin as an anti-inflammatoryagent in cases where inflammation was caused by purely chemical meansand no virus or bacteria were present or involved. F or this test theeffect of Xerosin was observed on guinea pigs -skin with respect to thenecrotic lesions produced by injections of pure turpentine. In thesetests five control and five treated animals were each injectedintradermally with 0.05, 0.02, and 0.01 ml. of turpentine and thediameters of the areas of induration Iwere measured 24 and 48 hourslater. Xerosin reduced considerably the size of the reactions. The areaof necrosis in control lesions was hemorrhagic, contracting, andsloughing; while in the animals treated with xero- `sin, the lesionremained bland, yellow, and uncontracted.

Tests have also been made of the eect of xerosin in suppressing tumordevelopment iu chickens, following inoculation with Rous sarcoma virus.ln these tests, daily injections of Xerosin prolonged the period ofincubation (that is, time `from infection to day of first appearance oftumor). In addition, xerosin was found to modify the gross appearance ofthe tumor in certain instances. Further, lmortality of the chickens wasdelayed.

Tests have shown, for example, that when Rous sarcoma Virus is injectedin relatively low concentrations into chicken wings, the tumorsresulting therefrom may be benecially affected by daily injections ofxerosin, whether those daily injections are started prior to theintroduction of the Rous sarcoma virus into the chickens or there` afterand upon the initial observation of a resulting tumor 3 mm. or more indiameter. It was found, for example, that lwhen chickens withestablished tumors were treated with xerosin, a Ivery large proportionof the tumors developed an atypical appearance as compared with typicaltumors. ln this respect these two types of tumors are distinguishable-as follows: Typical tumors were soft, grew rapidly, land were grosslyinvasive; while the xerosininduced atypical -tumors were hard, sharplycircumscribed, and grew slowly.

It was found that there was a striking similarity between the grossappearance of the atypical tumors induced by Xerosin and those inducedby a similar application to the host of hydrocortisone. However, thelatter promptly reverted to typical invasive tumors when the applicationof hydrocortisone was discontinued; while atypical tumors induced byxerosin continued to grow slowly, bu-t none .reverted to the typicalgrossly invasive type of tumor. As

a further distinction between the effects of the use of xerosin andhydrocortisone, it was found that when injections of hydrocortisone werebegun after inoculation of the chickens withRous sarcoma virus, thetumors were not only typical, but also grew even more rapidly thancontrol tumors.

Further, comparing the effects of the use of xerosin and cortisone(and/or a derivative of the latter, as `hydrocortisone) it has beenfound that in treating influenza virus infections in mice, injections ofXerosin are helpful, whereas injections of cortisone produce positiveundesirable eects. As to viral toxicity (instances where the virus isintroduced into an animal so that it does not grow or multiply, butstill causes a lesion), injections of Xerosin are helpful, Whereasinjections of cortisone have no appreciable eifect. In the case ofinfiamma-tions produced by chemicals, lfor example, turpentine or bybacterial endotoxins (for example, lipopolysaccharide) injections ofxerosin and cortisone are both helpful.

It has been shown above by a large number of tests on a considerablenumber of dierent representative viruses that xerosin is eective insuppressing the disease or lesion, due to or initiated by the presencein the host of the virus in question. lt has also been explainedgenerally that there is a growing body of evidence tending to show thatthe action of xerosin is selective on the disease or lesion, while thismaterial is ineffective as an antiviral agent, either as a virucidal oras a virustatic agent. As such, when a virus continues to multiply, theeffect of Xerosin is transient or temporary, delaying the progress ofthe lesion for perhaps 48 to 72 hours. It will be noted that Where thevirus is progressively multiplying and where xerosin can give a delayingaction only, there is incomplete and inadequate therapy.

What is, of course, desired is that the host shall recover promptly andcompletely from the disease in question. This recovery may in someinstances require the joint function of two or more agents in instanceswhere virus continues to multiply: first, something must be administeredto retard or suppress the disease, inflammation, or lesion-such asxerosin, and second, in those instances where viral multiplicationcontinues unabated throughout disease, it is also desirable to inhibitsuch viral multiplication. Based upon this reasoning, tests have beenmade using Xerosin jointly with previously known antibiotics which `alsoinhibit the multiplication of rickettsiae and certain larger virusesofthe psittacosis group, and particularly with such antibiotics aschlortetracycline and oxytetracycline fand also with chloramphenicol,all of which have a virustatic effect against mouse'pneumonitis virus.The action of these jointly administered therapeutic agents and theirseveral independent results, when used on mice infected with mousepneumonitis virus, is illustrated in FIGS. 8, 9 and l0. The therapeuticagents and the xerosin where used were, in each instance, injectedparenterally and the tests conducted in substantially the same manner ashas been more particularly described hereinabove. It will be noted thatthe conjoint effect of these 17 agents is, in many instances, to savethe life of the animal, which otherwise die under the conditions of thetest. It is believed that this is a new and useful result based uponthis combined therapy, which could not be attained by any one agentalone.

While it has been demonstrated that xerosin is effective in suppressingdisease or lesions resulting from t-he action of certain viruses incertain of the smaller laboratory animals (the mouse and the chicken,respectively), no ,reason is presently known Why this novel materialXerosin herein described will not eifect similar results in some of thelarger animals and perhaps also in human beings. Furthermore, while thepresent knowledge in regard to Xerosin is still somewhat limited,research Work in respect thereto is progressing and continuing, so thatfurther detailed infomation will become available as the workprogresses. The present disclosure embodies sub,- stantially all theinformation available to date in respect to this material, its method ofpreparation and its uses. Based upon the disclosure herein given, thoseskilled in the art could, and presumably will, lbe enabled to visualizeequivalents of certain of the procedures and of the substances hereinparticularly described. All such matter, reasonably equivalent to thatparticularly disclosed herein, should be considered as a part of thepresent invention which -is measured by the appended c1aims,whic\h areto be construed validly as broadly as the state of the prior artpermits.

What is claimed is:

1. A microbial product, Xerosin, which is active as an anti-inammatoryagent and is capable of benelicially atecting certain viral diseases;which is organic and contains nitrogen and phosphorus in compound form;which is not dialyzable against running water; which is soluble in water(pH=7) and in alkaline aqueous liquids; which is thermostable; which issubstantially insoluble in ethanol, n-butanol, anesthetic ether,chloroform, petroleum ether, ethyl acetate, benzene, and acetone; whichresponds as follows to certain color tests.

Test: Response Biuret Positive Anthrone Positive Iodine NegativeMolischs Negative Fehlings Negative Millons reagent; NegativeNitroprusside Negative Ninhydrin Y Negative and in vivo in suppressingthe development of the tumor in chickens previously infected with Roussarcoma virus; which is tolerated in said mice and chickens respectivelyin the concentrations required to suppress the lesions resulting fromthe actions of the respective viruses aforef said; which isdistinguished from cortisone and hydrocortisone in that it producesdesirable results in use in connection with inluenza virus infections inmice and virus-induced Rous sarcoma tumors in chickens, wherein suchresults do not ensue from the similar use of cortisone andhydrocortisone, and also has a benecial eiect on lesions resulting fromviral toxicity in mice, wherein cortisone is ineffective; and which isalso effective in reducing inamation induced by purely chemical meansand wherein neither virus nor bacteria are present as a cause of theinilammation; and which is derived from the cultivation of a strain ofAchromobacte xerosis No.134, and occurs in a crude state in theprecipitate from the culture 'medium of said Achromobaczer xeosis No.134 precipitated rby adjusting the pH thereof to a value from about 2 toabout 4, and said crude Xerosin being puriiied by repeated solution inan aqueous liquid and reprecipitation by adjusting the pH of such liquidto an acid condition of about 2 to about 4.

2. The method of producing Xerosin, comprising the steps of culturingAchromobacter xerosis No. 134, a strain of which is deposited in theculture collection of the 1nstitute of Microbiology of Rutgers, theState University, New Brunswick, New Jersey, precipitating crude Xerosinfrom the liquid culture by adjusting the pIH thereof to a value fromabout 2 to about 4, and purifying said crude Xerosin by repeatedsolution in an aqueous medium having a pH greater than about 4 andreprecipitation by adjusting the pH of the resulting solution to an acidcondition of about 2 to about 4.

3. The method of producing Xerosin according to claim 2, in which theculturing step is effected with the liquid culture stationary andsubstantially without agitation, a pellicle being formedvduring theculturing step, and the pellicle-forming material being eliminated fromsaid culture medium by a ltering step immediately following theculturing step and prior to the initial precipitation of said crudeXerosin.

4. The method of producing Xerosin according to claim 2, in which saidpurifying step is effected by a reprecipitation of the Xerosin byadjusting the pH of each said resulting solution to about 3.5.

5. The method of producing Xerosin according to claim 2, in which boththe initial precipitation of said crude Xerosin and the repeatedreprecipitations included in said purification step are effected -byadjusting the pH of the respective solutions to about 3.5

References Cited in the le of this patent UNITED STATES PATENTS2,239,345 Sperti -..f Apr. 22, 1941 FOREIGN PATENTS 505,256 Belgium Aug.3l, 1951 OTHER REFERENCES Group etral.: J. Bact., vol. 68, pp. 10-18,1954.

Groupe et al.: =P.S.KE.\B.M., vol. 8, pp. 710, 1952.

Ginsberg: Fed. lProc., vol. 13, p. 494, 1954.

Groupe et al.: l. Immunology, vol. 74, p. 249, 1955.

Ricks et al.: Science, Dec. 3, 1948, pp. 634, 635.

Karel: Dictionary of Antibiotics, page 4, publ. 1951, ColumbiaUniversity Press, New York City.

Todd: l. Pharm. and Pharm., October 1955, pp. 6-25- 641.

1. A MICROBIAL PRODUCT, ZEROSIN, WHICH IS ACTIVE AS AN ANTI-INFLAMMATORYAGENT AND IS CAPABLE OF BENEFICIALLY AFFECTING CERTAIN VIRAL DISEASES;WHICH IS ORGANIC AND CONTAINS NITROGEN AND PHOSPHORUS IN COMPOUND FORM;WHICH IS NOT DIALYZABLE AGAINST RUNNING WATER; WHICH IS SOLUBLE IN WATER(PH=7) AND IN ALKALINE AQUEOUS LIQUIDS; WHICH IS THERMOSTABLE; WHICH ISSUBSTANTIALLY INSOLUBLE IN ETHANOL, N-BUTANOL, ANESTHETIC ETHER,CHLOROFORM, PETROLEUM ETHER, ETHYL ACETATE, BENZENE, AND ACETONE; WHICHRESPONDS AS FOLLOWS TO CERTAIN COLOR TESTS. BIURET POSITIVE ANTHRONEPOSITIVE IODINE NEGATIVE MOLISCH''S NEGATIVE FEHLING''S NEGATIVEMILLON''S REAGENT NEGATIVE NITROPRUSSIDE NEGATIVE NINHYDRIN NEGATIVEWHICH HAS AN ULTRAVIOLET ABSORPTION PATTERN SUBSTANTIALLY AS SHOWN INFIG. 1, WITH A PEAK AT ABOUT 255-260 MU; WHICH IS SUBSTANTIALLYINEFFECTIVE AS A VIRUCIDAL AGENT IN VITRO OR AS A VIRUSTATIC AGENT INVIVO; WHICH IS EFFECTIVE IN VIVO IN SUPRESSING THE DEVELOPMENT OFLEISONS IN MICE PREVIOUSLY INOCULATED WITH ANY ONE OF THE FOLLOWINGVIRUSES: FINLUENZA A VIRUS INFLUENZA B VIRUS MOUSE PNEUMONITIS VIRUS(MIYAGAWANELLA BRONCHOPNEUMONIAE) NEWCASTLE DIESASE VIRUS AND IN VIVO INSUPPRESSING THE DEVELOPEMNT OF THE TUMOR IN CHICKENS PREVIOUSLY INFECTEDWITH ROUS'' SARCOMA VIRUS; WHICH IS TOLERATED IN SAID MICE AND CHICKENSRESPECTIVELY IN THE CONTENTRATIONS REQUIRED TO SUPPRESS THE LESIONSRESULTING FROM THE ACTIONS OF THE RESPECTIVE VIRUSES AFORESAID; WHICH ISDISTINGUISHED FROM CORTISONE AND HYDROCORTISONE IN THAT IT PRODUCESDESIRABLE RESULTS IN USE IN CONNECTION WITH INFLUENZA VIRUS INFECTIONSIN MICH AND VIRUS-INDUCED ROUS'' SARCOMA TUMORS IN CHICKENS, WHEREINSUCH RESULTS DO NOT ENSURE FROM THESIMILAR USE OF CORTISONE ANDHYDROCORTISONE, AND ALSO HAS A BENEFICIAL EFFECT ON LESIONS RESULTINGFROM VIRAL TOXICITY IN MICE, WHEREIN CORTISONE IS INEFFECTIVE; AND WHICHIS ALSO EFFECTIVE IN REDUCING INFLAMATION INDUCED BY PURELY CHEMICALMEANS AND WHEREIN NEITHER VIRUS NOR BACTERIA ARE PRESENT AS A CAUSE OFTHE INFLAMMATION; AND WHICH IS DERIVED FROM THE CULTIVATION OF A STRAINOF ACHROMOBACTER XEROSIS NO.134, AND OCCURS IN A CRUDE STATE IN THEPRECIPITATE FROM THE CULTURE MEDIUM OF SAID ACHROMOBACTER XEROSIS NO.134 PRECIPITATED BY ADJUSTING THE PH THEREOF TO A VALUE FROM ABOUT 2 TO4, AND SAID CRUDE EXEROSIN BEING PURIFIED BY REPEATED SOLUTION IN ANAQUEOUS LIQUID AND REPRECIPITATION BY ADJUSTING THE PH OF SUCH LIQUID TOAN ACID CONDITION OF ABOUT 2 TO ABOUT 4.